Over the past year, we have published several components of the specific aims in the original proposal and have extended our investigations to discern how HIV could be activating microglia in an effort to identify better therapeutic compounds. Specifically, we have: A) Identified the smallest peptide fragment of dynorphin required for anti-inflammatory and neuroprotective effects. Relevant recent publications are: (Qin et. al, FASEB J 2005; Qin et al., Annals of the New York Academy of Sciences, in press). After testing several peptide fragments of dynorphin, it was determined that any sequence containing gly-gly-phe (GGF) was both anti-inflammatory and neuroprotective. We show that both gly-gly-phe (GGF), a tri-peptide contained in the dynorphin opioid peptide, and naloxone are neuroprotective at femtomolar concentrations against LPS-induced dopaminergic neurotoxicity through the reduction of microglial activation. Mechanistic studies demonstrated the critical role of NADPH oxidase in the GGF and naloxone inhibition of microglial activation and associated DA neurotoxicity. Previously, we have identified that NADPH oxidase contributes to neurotoxicity through two mechanisms. First, activation of NADPH oxidase results in the production of neurotoxic ROS. Second, NADPH oxidase activation increases intracellular ROS in the phagocyte, resulting in amplification of pro-inflammatory factors. However, in the case of HIV-associate dementia (HAD) therapy, attenuating NADPH oxidase may provide an additional benefit of reducing the activation of the LTR promoter, helping to attenuate the vicious cycle of microglial activation. Further, the targeting of NADPH oxidase may allow a selective inhibition of the activation of phagocytic cells, a characteristic of great importance to an immune system that is already comprised with T cell immunodeficiency. B) Identified other small molecules with neuroprotective mechanisms similar to dynorphin that are better able to traverse the blood brain barrier. Relevant recent publications are: (Li et al., FASEB J 2005). Dextromethorphan, Naloxone, morphine, sinomalin and diphenyl iodonium (DPI), an NADPH oxidase inhibitor, have all been shown to be neuroprotective through the inhibition of microglial NADPH oxidase at both micromolar and femtomolar concentrations. C) Identified a novel mechanism of microglia-mediated HIV-associated neurotoxicity, where gp120 induces microglia derived oxidative stress. The HIV viral coat protein ,gp120, is shed by microglia and has been reported to be directly toxic to neurons and to activate microglia, which may provide a self propelling cycle of microglial activation. However, while chemokine receptors have been named as critical to the process of GP120 activation, the mechanisms of GP120-induced microglial activation remains undefined. Recently, we have identified several factors that activate microglia through receptors involved in the phagocytosis of aggregated proteins (a synuclein, neuromelanin) or environmental particulates. These toxic compounds are recognized by phagocytosis/pattern recognition receptors on microglia, resulting in the activation of NADPH oxidase and the neurotoxic respiratory burst. Recently, we have shown that at nanomolar concentrations, gp120 is toxic to neurons only the presence of microglia and that gp120 produces extracellular and intracellular reactive oxygen species from microglia in response to LPS. Further, using neuron-glia cultures from mice lack a functional NADPH oxidase complex, we show that NADPH oxidase and extracellular superoxide plays a critical role in the microglia-mediated GP120-induced DA neurotoxicity. While it is not yet conclusive whether gp120 is being phagocytized and whether this is critical for microglial activation, further efforts in our laboratory will attempt to elucidate the mechanisms responsible for this phenomenon. Through identification of the mechanisms driving the neurodegeneration associated with HAD, we can hope to have better insight into the development of better therapeutic compounds.